An isotopic method was developed to measure the in vivo rate of dephosphorylation of glucose 6-phosphate in brains of conscious rats. A mixture of [2-3H,U-14C] glucose was injected into the left internal carotid artery and the change in the 3H/14C ratio of cerebral glucose was examined in freeze-blown brains at various times following injection. The 3H/14C ratio in cerebral glucose decreased approximately 35% in the 5 min following injection. During the same period the isotopic ratio in blood glucose decreased less than 5%. This indicates that the decrease in 3H/14C ratio in cerebral glucose was caused by the dephosphorylation of glucose 6-phosphate in cerebral tissues, not by uptake of the recirculated labeled glucose by brain. The rate of dephosphorylation of glucose 6-phospate was calculated by subtracting the rate of phosphorylation of [U-14C] glucose from that of [2-3H] glucose. The rate of phosphorylation (F) of labeled glucose was calculated as follows. F= (disintegration/min in metabolic products of glucose/g of tissue)/(Specific activity of glucose (dpm.umol-1).time (min)). The extrapolated rate of flux through glycolysis measured with [U-14C] glucose was 0.86 umol.min-1.g-1. The rate of dephosphorylation was estimated as 0.30 umol.min-1.g-1, or about 35% of the rate of glucose utilization. The 3H/14C ratio in cerebral glucose from rats injected with [6-3H, U-14C] glucose did no show significant changes in 10 min, indicating further that the dephosphorylation of glucose 6-phosphate observed in the experiments with [2-3H, U-14C] glucose was not the result of recirculation of label into brain from peripheral tissues or the reversal of the entire glycolytic pathway.